Second Generation Fatty Acid Compositions, Formulations, and Methods of Use and Synthesis Thereof

ABSTRACT

An orally administered fatty acid composition for the treatment of cardiovascular diseases, and a method of treating same, are provided. The compound includes 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic ethanolamide (EPA ethanolamide), 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic ethanolamide (DHA ethanolamide), and at least one tocotrienol. The EPA ethanolamide and the DHA ethanolamide are preferably each substantially in a range of 100-900 mg per dosage form. The at least one tocotrienol is substantially in a range of 10-500 mg per dosage form. The at least one tocotrienol includes at least one of α-tocotrienol, β-tocotrienol, γ-tocotrienol, or δ-tocotrienol and is preferably substantially tocopherol-free. The composition may take the form of a medical food or a pharmaceutical preparation. A preferred formulation of the composition includes approximately 525 mg EPA ethanolamide, approximately 315 mg DHA ethanolamide, and approximately 50 mg δ-tocotrienol. The EPA and DHA ethanolamides may be synthesized from fatty acid triglycerides.

RELATED APPLICATIONS

Domestic priority is claimed from U.S. Provisional Patent ApplicationNo. 61/232,554 entitled “Second Generation Fatty Acid Compositions,Formulations, and Methods of Use and Synthesis Thereof,” filed Aug. 10,2009, and from U.S. Provisional Patent Application No. 61/288,587entitled “Second Generation Fatty Acid Compositions, Formulations, andMethods of Use and Synthesis Thereof,” filed Dec. 21, 2009 theentireties of both of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method utilizing a singleadministration or a unit dosage of omega-3 fatty acids and/or omega-3fatty acid amides for the treatment of patients with dyslipidemias,including hypertriglyceridemia, high LDL-C serum levels, coronary heartdisease (CHD), vascular disease, artherosclerotic disease, hypertension,and related conditions, and the prevention or reduction ofcardiovascular and vascular events.

2. Description of Related Art

Cardiovascular diseases leading to morbidity and premature mortality arerelated to several risk factors such as hypertension,hypertriglyceridemia, hypercholesterolemia, high blood plateletaggregation and according to recent findings, a high activity of theblood coagulation factor VII phospholipid complex. Over the last fourdecades, antihypertensive drugs have contributed to the decline incardiovascular disease-related morbidity and mortality. There is,however, heightened concern about side effects and toxicity associatedwith the current antihypertensive therapy, especially in the mildlyhypertensive patient. There are results indicating that, although someantihypertensive agents are efficient in reducing blood pressure, thepulse rate is coincidentally increased. Thus, there is a need for a drugwith fewer adverse effects for the treatment of hypertension. It wouldbe particularly advantageous if such a drug could be used for thesimultaneous treatment of all the above mentioned multiple risk factorsassociated with cardiovascular diseases, which is generally not the casewith the currently available antihypertensive drugs.

During the late 1980s through the mid-1990s, numerous publicationsappeared which report that various dietary fish oil preparationscontaining omega-3 polyunsaturated fatty acids have the effect oflowering serum triglycerides and cholesterol.

There are currently over 40 different non-prescription, over-the-counterproducts containing omega-3 fatty acids, usually as triglycerides. Inthese formulations, the triglycerides are polydisperse, containing bothsaturated as well as unsaturated fatty acids. For example, flax seed oilhas a typical composition as follows:

Mono Poly Saturated unsaturated unsaturated Unsat./ Capric LauricMyristic Palmitic Stearic Oleic Linoleic Alpha Linolenic Sat. Acid AcidAcid Acid Acid Acid Acid (ω6) Acid (ω3) Oil or Fat ratio C10:0 C12:0C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 Flaxseed Oil 9.0 — — — 3 7 21 16 53In some cases the saturated fatty acids can interfere with the effectsof the polyunsaturated omega-3 fatty acids.

U.S. Pat. Nos. 5,502,077 and 5,656,667, both to Breivik et al., describea lipid-regulating agent, as a liquid-filled gel capsule for oraladministration. Each 1-gram capsule of this formulation, which iscommercialized as Lovaza® (GSK, Research Triangle Park, N.C. 27709),contains at least 900 mg of the ethyl esters of omega-3 fatty acids.These are predominantly a combination of ethyl esters ofeicosapentaenoic acid (EPA—approximately 465 mg) and docosahexaenoicacid (DHA—approximately 375 mg).

In patients with very high triglyceride levels (>500 mg/dL) Lovaza isreported to lower triglyceride levels 44.9% [Lovaza Package Insert,Table 2]. Unfortunately this formulation adversely affects low densitylipoprotein cholesterol (LDL-C), increasing it by 44.5%. Patients are atan increased risk of cardiovascular diseases and events when their LDL-Cincreases.

There is therefore a need to provide the beneficial effects of omega3-fatty acids in compositions and formulations that do not have theLDL-C raising effects of the prior art formulations but in fact lowerboth LDL-C and triglycerides.

SUMMARY OF THE INVENTION

It has been found that fatty acid compositions containing omega-3 fattyacid amides (rather than their ethyl esters), including the ethanolamineamides, in an acceptable formulation, not only lower abnormally hightriglycerides but also cholesterol, in the form of LDL-C. Accordingly, afirst aspect of the invention is a method of treating high triglycerideand high LDL-C blood levels in a mammalian patient. A therapeuticallyeffective amount of omega-3 fatty acids and/or omega-3 fatty acid amidesis administered in an acceptable carrier sufficient to lower bothtriglyceride and LDL-C blood levels. Preferably, the omega-3 fatty acidsand/or omega-3 fatty acid amides include at least one ofoctadecatrienoic (ALA), eicosapentaenoic (EPA), or docosahexenoic (DHA)acid or ethanolamide. More preferably, the composition includes both EPAand DHA. These compositions can also be formulated with otherlipid-lowering compounds, including statins, niacin-containingcompounds, cholesterol-absorbing blockers, ethanolamine, and/or anHMG-CoA reductase inhibitor, including one or more tocotrienols.

In another aspect of the invention, the invention includes a method ofsynthesizing omega-3 fatty acid ethanolamides from fatty acidtriglycerides. While the synthesis of fatty acid ethanolamides from theethyl esters of EPA and DHA derived from plant sources (flax seed) andmackerel pike (Cololabis sair) has been described, the synthesis fromomega-3 fatty acid triglycerides has not been described.

In one aspect of the invention, an orally administered fatty acidcomposition includes 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic ethanolamide(EPA ethanolamide), 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic ethanolamide(DHA ethanolamide); and at least one tocotrienol. The composition has atotal aerobic plate count per g/mL of less than 20,000 cfu. Preferably,the EPA ethanolamide is substantially in a range of 100 mg to 900 mg perdosage form, the DHA ethanolamide is substantially in a range of 100 mgto 900 mg per dosage form, and the at least one tocotrienol issubstantially in a range of 10 mg to 500 mg per dosage form.

The at least one tocotrienol includes at least one of α-tocotrienol,β-tocotrienol, γ-tocotrienol, or δ-tocotrienol and is preferablysubstantially tocopherol-free.

The composition may be a medical food, a pharmaceutical preparation, oranother oral formulation.

Preferably, the orally administered fatty acid composition of theinvention includes a capsule having approximately 525 mg EPAethanolamide, approximately 315 mg DHA ethanolamide, and approximately50 mg δ-tocotrienol. The capsule preferably has a gelatin base.Additionally, at least one of the EPA ethanolamide or the DHAethanolamide may be synthesized from a respective fatty acidtriglyceride.

In another aspect of the invention, a method of treating cardiovasculardiseases includes the step of orally administering a therapeuticallyeffective amount of a composition having5Z,8Z,11Z,14Z,17Z-eicosapentaenoic ethanolamide (EPA ethanolamide),4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic ethanolamide (DHA ethanolamide),and at least one tocotrienol. Preferably, The EPA ethanolamide issubstantially in a range of 100-900 mg per dosage form, the DHAethanolamide is substantially in a range of 100-900 mg per dosage form,and the at least one tocotrienol is substantially in a range of 10-500mg per dosage form. The at least one tocotrienol includes at least oneof α-tocotrienol, β-tocotrienol, γ-tocotrienol, or δ-tocotrienol and ispreferably substantially tocopherol-free. The orally administeredcomposition may be a medical food, a pharmaceutical preparation, orother oral formulation.

Optionally, the composition comprises a capsule having approximately 525mg EPA ethanolamide, approximately 315 mg DHA ethanolamide, andapproximately 50 mg δ-tocotrienol. The EPA ethanolamide, the DHAethanolamide, and the at least one tocotrienol may be provided in acapsule having a gelatin base. The method may optionally include thestep of synthesizing at least one of the EPA ethanolamide or the DHAethanolamide from a respective fatty acid triglyceride.

DEFINITIONS

n-3 fatty acids (popularly referred to as ω-3 fatty acids or omega-3fatty acids) are a family of unsaturated fatty acids that have in commona final carbon-carbon double bond in the n-3 position; that is, thethird bond from the methyl end of the fatty acid.

This table lists several different names for the most common n-3 fattyacids found in nature.

TABLE I Lipid Common name name Chemical name 16:3all-cis-7,10,13-hexadecatrienoic acid (n-3) α-Linolenic 18:3all-cis-9,12,15-octadecatrienoic acid acid (ALA) (n-3) Stearidonic 18:4all-cis-6,9,12,15-octadecatetraenoic acid acid (STD) (n-3)Eicosatrienoic 20:3 all-cis-11,14,17-eicosatrienoic acid acid (ETE)(n-3) Eicosatetraenoic 20:4 all-cis-8,11,14,17-eicosatetraenoic acidacid (ETA) (n-3) Eicosapentaenoic 20:5all-cis-5,8,11,14,17-eicosapentaenoic acid (EPA) (n-3) acidDocosapentaenoic 22:5 all-cis-7,10,13,16,19-docosapentaenoic acid (DPA),(n-3) acid Clupanodonic acid Docosahexaenoic 22:6all-cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) (n-3) acidTetracosapentaenoic 24:5 all-cis-9,12,15,18,21-docosahexaenoic acid(n-3) acid Tetracosahexaenoic 24:6 all-cis-6,9,12,15,18,21-tetracosenoicacid (Nisinic acid) (n-3) acid

DETAILED DESCRIPTION OF THE INVENTION

A more detailed description of the invention will now be given. Itshould be understood that this description is exemplary in nature and inno way serves to limit the scope of the invention, which is defined bythe claims appearing hereinbelow.

The invention includes a preferably oral formulation (e.g., apharmaceutical preparation or a medical food) of omega-3 fatty acidamides, including the ethanolamine amides. Unlike the ethyl esters orthe triglycerides of omega-3 fatty acids, the amides have severalbeneficial effects without many of the concomitant negative effects.First, the amides of omega-3 fatty acids boost production ofphospholipids by, among other reasons, contributing both theethanolamine head group and the fatty acid chains tophosphatidylethanolamine and phosphatidylcholine. Second, they aremetabolized into substances that are typically in short supply in thehuman body: ethanolamine and omega-3 fatty acids. Further, amides ofomega-3 fatty acids should not raise LDL cholesterol levels as do theethyl esters and triglycerides of omega-3 fatty acids. Indeed, in thepreferred embodiment, when combined with one or more tocotrienols,omega-3 fatty acid amides should advantageously lower both triglyceridesand LDL cholesterol. In the alternative to providing an oral formulationof the amides of omega-3 fatty acids, the invention also includes anoral formulation of omega-3 fatty acids and ethanolamine, preferablyalso including at least one tocotrienol.

Below are described exemplary and prophetic processes for synthesizingomega-3 fatty acid amides.

1. Lipid Extraction

Lipids can be extracted from flax seed and saira carcasses according tothe literature method [E. G. Bligh and W. J. Dyer, Can. J. Biochem.Physiol., 37, No. 8, 911 (1959).].

2. Synthesis of FA Ethanolamides from FA Ethyl Esters

The method of Karaulov et. al. (Synthesis of Fatty-Acid Ethanolamidesfrom Linum catharticum Oils and Cololabis sairsa Fats, Chemistry ofNatural Compounds, Vol. 40, No. 3, 2004), repeated here, can be used. Inthe reference, a mixture of FA ethyl esters (6.56 g) was treated withfreshly distilled monoethanolamine (over KOH, 6.53 g, 1:5 mole ratio)and trifluoroacetic acid (10 μL). The mixture was sealed in an ampul,vigorously shaken, held for 2 h at 140° C., cooled to 25° C., andremoved from the ampul. The contents were treated with CHCl₃ (10 mL) andaqueous HCl (10%) until the pH was 3. The resulting mixture wasvigorously shaken. The CHCl₃ layer was separated after layering. Theextraction was repeated twice. The CHCl₃ fractions were combined, driedover anhydrous Na₂SO₄, and evaporated in vacuum at 40° C. to constantmass. Yield 6.67 g (6.54 g for FA ethyl esters from flax-seed oil) as anopaque yellowish-orange oil containing 4.86 g (4.78 g for FA ethylesters from flax-seed oil) of FA ethanolamides. The degree of conversionwas about 70%. The purity of the FA ethanolamides was monitored by TLC.The FA ethanolamides were purified over a Chemapol L silica-gel column(Kavalier, Czech Rep., 40-100 μm) using a stepped gradient of acetone inhexane (0→10%) calculated as 2 g of starting ethanolamides per 60 gsilica gel. Yield 900 mg of purified FA ethanolamides (Rf 0.47) fromsaira fat and flax-seed oil as colorless oily liquids for every 2 g ofstarting mixture placed on the column. An analogous procedure wasperformed using ethyl esters of α-linolenic, eicosapentaenoic, anddocosahexaenoic acids to afford FA ethanolamides of these FA (Rf 0.47)as oily liquids in yields of 33, 34, and 35 mg, respectively, for each100 mg of FA ethyl esters used in the reaction. All FA ethanolamidesfrom pure FA ethyl esters and from mixtures were analyzed by HPLC-MS(APCI).

3. Prophetic Synthesis of FA Ethanolamides from FA Triglycerides

In accordance with the invention, FA ethanolamides can prophetically besynthesized directly from FA triglycerides, rather than from FA ethylesters as described above. Specifically, the invention includes a methodin which FA ethanolamides are to be prepared by aminolysis of the threeester bonds of the FA triglycerides by three mole equivalents ofethanolamine. FA triglycerides are typically much less expensive than FAethyl esters, and the overall process would entail far fewer steps thanthat described above. As such, the prophetic inventive method isexpected to be less expensive, more convenient, and faster than previousmethods.

FA triglycerides (6.56 g) can be treated with freshly distilledmonoethanolamine (over KOH, 6.53 g, 1:5 mole ratio) and trifluoroaceticacid (10 μL). The mixture can be sealed in an ampul, vigorously shaken,held for 2 h at 140° C., cooled to 25° C., and removed from the ampul.The contents could be treated with CHCl₃ (10 mL) and aqueous HCl (10%)until the pH reaches 3. The resulting mixture could be vigorouslyshaken. The CHCl₃ layer could then be separated after layering. Theextraction could be repeated twice. The CHCl₃ fractions could becombined, dried over anhydrous Na₂SO₄, and evaporated in vacuum at 40°C. to constant mass. A yield of 6.67 g (6.54 g for FA triglycerides fromflax-seed oil) as an opaque yellowish-orange oil containing 4.86 g (4.78g for FA triglycerides from flax-seed oil) of FA ethanolamides could befound. The degree of conversion is expected to be about 70%. The purityof the FA ethanolamides can be monitored by TLC. The FA ethanolamidescan be purified over a Chemapol L silica-gel column (Kavalier, CzechRep., 40-100 μm) using a stepped gradient of acetone in hexane (0→10%)calculated as 2 g of starting ethanolamides per 60 g silica gel. A yield900 mg of purified FA ethanolamides (Rf 0.47) from saira fat andflax-seed oil as colorless oily liquids for every 2 g of startingmixture placed on the column could be expected. An analogous procedurecould be performed using triglycerides of α-linolenic, eicosapentaenoic,and docosahexaenoic acids to afford FA ethanolamides of these FA (Rf0.47) as oily liquids in yields of 33, 34, and 35 mg, respectively, foreach 100 mg of FA triglycerides used in the reaction. All FAethanolamides from pure FA triglycerides and from mixtures could beanalyzed by HPLC-MS (APCI).

4. Exemplary Prophetic Preparation

One exemplary prophetic preparation could be as follows:

Soft gelatin capsules containing 1 g/per capsule

Composition:

EPA ethanolamide 525 mg/capsuleDHA ethanolamide 315 mg/capsuleDelta-tocotrienol 50 mg/capsuleGelatine 246 mg/capsuleGlycerol 118 mg/capsuleRed iron oxide 2.27 mg/capsuleYellow iron oxide 2.27 mg/capsule

The active ingredients and the excipients can be weighed and homogenizedon a high speed stirrer. The mixture can then be then colloid milled andde-aerated in a stainless steel vessel ready for encapsulation. Themixture can be filled in soft gelatin capsules of size 20 oblong(average weight 1.4 g) using a standard capsulation machine. It ispreferred that the delta-tocotrienol be substantially tocopherol-free.The tocotrienol (or TCT) group—together with tocopherols—compose thevitamin E family. Natural tocotrienols exist in four different forms orisomers, named alpha-, beta-, gamma- and delta-tocotrienol, each whichcontain different number of methyl groups on the chromanol ring. Themajor structural difference from tocopherol is through its unsaturatedside chain that has three double bonds in its farnesyl isoprenoid tail.In the above example, the delta-tocotrienol can be replaced oraccompanied with portions of the other three tocotrienols. The aboveexample can be prepared as a medical food, a pharmaceutical preparation,or other variations of preparations that are ingestible by oradministrable to a mammal.

5. Standards and Testing for Microbial Contamination of the Oral SolidDosage Form of the Instant Inventive Composition

The publication, “Microbial Bioburden on Oral Solid dosage Forms,” byJose E. Martinez, Pharmaceutical Technolgy, February 2002, pages 58 to70, is incorporated herein by reference. For formulations of the instantinventive composition, the water activity is 0.85, testing for TAC andUSP indicator organisms is not necessary.

Furthermore, since formulations of the instant inventive compositionalso have water activity of 0.75, then no detailed microbiologicaltesting of that product should be done.Total aerobic plate count (TAC) is an estimation of the total viableaerobic bacteria present in a sample of raw material, in-processmaterial, or finished product. Samples are analyzed in accordance withthe most current USP Guidelines Chapter <61>, Microbial Limits Test.Acceptable Total aerobic plate colonies (TAC) for OSDFs are establishedfor the formulations of the inventive composition in terms of alert andaction levels, which could be 1000 cfu g/mL and 10,000 cfu g/mL,respectively. A TAC that is 20,000 cfu g/mL is unacceptable.

6. Testing of Formulations of the Instant Inventive Composition

The effects of the prior art formulations of U.S. Pat. No. 5,656,667(LOVAZA) 4 g per day and of this invention can be assessed inrandomized, placebo-controlled, double-blind, parallel-group studies of84 adult patients (42 on LOVAZA, 42 on placebo) with very hightriglyceride levels (Table 2). Patients whose baseline triglyceridelevels are between 500 and 2,000 mg/dL can be enrolled in these studieswhich can have a 6 and 16 weeks' duration. The median triglyceride andLDL-C levels in these patients can be approximately 792 mg/dL and 100mg/dL, respectively. Median HDL-C level can be about 23.0 mg/dL.

The changes in the major lipoprotein lipid parameters for the groupsreceiving LOVAZA or the changes expected from the exemplary propheticformulation above are shown in Table II.

TABLE II Parameter Lovaza Change Exemplary Formulation ChangeTriglyceride Decrease 45% Expected Decrease about 45% LDL-CholesterolIncrease 45% Expected Decrease about 20%

The invention is not limited to the above description. For example, inthe exemplary prophetic formulation, delta-tocotrienol is included withthe omega-3 fatty acid amides. However, any combination of alpha-,beta-, gamma, and/or delta tocotrienol may also be included. Further,while amides of DHA and EPA are used in the exemplary propheticformulation, amides of other omega-3 fatty acids (e.g., selected fromthe list appearing on Table I above) may be included in addition or inthe alternative. While the ethanolamide amides of the omega-3 fattyacids are described, any other amine, whether primary or secondary,aliphatic or aromatic, can be substituted. Additionally, while specificamounts of the amides of DHA and EPA are listed in the exemplaryprophetic formulation above, the invention is not so limited; rather,substantially any therapeutically effective amount of these or otheramides of omega-3 fatty acids may be incorporated into the formulation.

Also, as mentioned above, the inventive oral formulation may includeomega-3 fatty acids and ethanolamine directly instead of omega-3 fattyacid amides (which would metabolize into omega-3 fatty acids andethanolamine). At least one tocotrienol is preferably included in thisformulation.

Moreover, other additives, excipients, and the like may be added or usedin the alternative. The actual commercialized product need not be agelatin-based capsule; “dry” and vegetarian formulations may also beemployed. Other variations are also contemplated. For example, theinventive composition should be orally administered in the form ofpills, soft capsules or the like. However, the administration could alsobe through any other route where the active ingredients may beefficiently absorbed and utilized, e.g. intravenously, subcutaneously,rectally, vaginally, or possibly topically.

Having described certain embodiments of the invention, it should beunderstood that the invention is not limited to the above description.Rather, the scope of the invention is defined by the claims appearinghereinbelow and any equivalents thereof as would be appreciated by oneof ordinary skill in the art.

1-11. (canceled)
 12. A method of treating cardiovascular diseases,comprising the step of orally administering a therapeutically effectiveamount of a composition comprising 5Z, 8Z, 11Z, 14Z,17Z-eicosapentaenoic ethanolamide (EPA ethanolamide), 4Z, 7Z, 10Z, 13Z,16Z, 19Z-docsahexaenoic ethanolamide (DHA ethanolamide), and at leastone tocotrienol.
 13. A method of treating cardiovascular diseasesaccording to claim 12, wherein the EPA ethanolamide is substantially ina range of 100-900 mg per dosage form, the DHA ethanolamide issubstantially in a range of 100-900 mg per dosage form, and the at leastone tocotrienol is substantially in a range of 10-500 mg per dosageform.
 14. A method of treating cardiovascular diseases according toclaim 12, wherein at least one tocotrienol includes at least one ofa-tocotrienol, 13-tocotrienol, y-tocotrienol, or 6-tocotrienol and issubstantially tocopherol-free.
 15. A method of treating cardiovasculardiseases according to claim 12, wherein the composition comprises amedical food.
 16. A method of treating cardiovascular diseases accordingto claim 12, wherein the composition comprises a pharmaceuticalpreparation.
 17. A method of treating cardiovascular diseases accordingto claim 12, wherein the composition comprises a capsule havingapproximately 525 mg of the EPA ethanolamide, approximately 315 mg ofthe DHA ethanolamide, and approximately 50 mg of 6-tocotrienol.
 18. Amethod of treating cardiovascular diseases according to claim 12,further comprising the step of provising the EPA ethanolamide, the DHAethanolamide, and the at least tocotrienol in a capsule having a gelatinbase.
 19. A method of treating cardiovascular diseases according toclaim 12, further comprising the step of synthesizing at least of saidEPA ethanolamide or said DHA ethanolamide from a respective fatty acidtriglyceride.